Analysis of amino acid copolymer compositions

ABSTRACT

Methods for analyzing, selecting, characterizing or classifying compositions of a co-polymer, e.g., glatiramer acetate are described. The methods entail analysis of pyro-glutamate in the composition, and, in some methods, comparing the amount of pyro-glutamate present in a composition to a reference standard.

RELATED APPLICATION INFORMATION

This application is a continuation of U.S. patent application Ser. No.13/709,586, filed Dec. 10, 2012, which is a continuation of U.S. patentapplication Ser. No. 12/408,058, filed Mar. 20, 2009, and claimspriority to U.S. Provisional Application Ser. No. 61/045,465, filed Apr.16, 2008. The entire disclosures of the prior applications areconsidered part of, and are incorporated by reference in theirentireties in, the disclosure of this application.

BACKGROUND

Glatiramer acetate (also known as copolymer-1 and marketed as the activeingredient in COPAXONE® by Teva Pharmaceutical Industries Ltd., Israel)is used in the treatment of the relapsing-remitting form of multiplesclerosis (RRMS). According to the COPAXONE® product label, glatirameracetate (GA) consists of the acetate salts of synthetic polypeptides,containing four naturally occurring amino acids: L-glutamic acid,L-alanine, L-tyrosine, and L-lysine with a reported average molarfraction of 0.141, 0.427, 0.095, and 0.338, respectively. Chemically,glatiramer acetate is designated L-glutamic acid polymer with L-alanine,L-lysine and L-tyrosine, acetate (salt). Its structural formula is:

(Glu, Ala, Lys, Tyr)_(x).xCH₃COOH

(C₅H₉NO₄.C₃H₇NO₂.6C₆H₁₄N₂O₂.C₉H₁₁NO₃)_(x) .xC₂H₄O₂

CAS-147245-92-9 SUMMARY OF THE INVENTION

The invention is based, at least in part, on the identification andcharacterization of L-pyroGlutamic Acid (gyro-Glu) as a structuralsignature of glatiramer acetate (GA). Analysis of this signaturecomponent of GA is useful to assess product and process quality in themanufacture of GA.

Described herein is a method of selecting a batch of a compositioncomprising an amino acid copolymer (e.g., GA), the method comprising:providing a batch of a composition comprising an amino acid copolymer;measuring the amount of gyro-glutamate (pyro-Glu) in the batch; andselecting the batch if the amount of pyro-Glu in the batch is within apredetermined range. In this method, as in the other methods describedherein, the measuring step can employ any suitable method and the unitsused to express the measured amount of pyro-Glu can be any suitableunits (e.g., ppm or mole percent of chains). In measuring the amount ofpyro-Glu, one can, e.g., measure the concentration of pyroGlu in asample or the total amount of pyro-Glu in sample. However an amount ofpyro-Glu is measured and whatever units are used to express the measuredamount, the concentration of pyro-Glu in the selected batch is between2000 and 7000 ppm (in some cases between 2500 and 6500 ppm) on a dryweight/dry weight basis.

Also described is a method for preparing a pharmaceutical compositioncomprising: providing a batch of a composition comprising an amino acidcopolymer, measuring the amount of pyro-Glu in the batch; and preparinga pharmaceutical composition comprising at least a portion of the batchif the amount of pyro-Glu in the batch is within a predetermined range.Here too, the measuring step can employ any suitable method and unitsused to express the measured amount of pyro-Glu can be any suitableunits (e.g., ppm or mole percent of chains). However the pyro-Glu ismeasured and whatever units are used to express the measured amount, theconcentration of pyro-Glu in the selected batch is between 2000 and 7000ppm (in some cases between 2500 and 6500 ppm) on a dry weight/dry weightbasis.

A batch of a composition comprising an amino acid copolymer can be allor part of the product of a copolymer manufacturing process (e.g., allor part of a single manufacturing run). In some cases, one batch isanalyzed. In some cases two or more batches are analyzed. In some cases,multiple samples taken from a single batch are analyzed. The compositioncontaining a copolymer can be a drug substance (DS) (also known as anactive pharmaceutical ingredient (API)), a drug product (DP), or aprocess intermediate. The copolymer can be glatiramer acetate.

Also described is a method for preparing a pharmaceutical compositioncomprising glatiramer acetate, comprising: polymerizing N-carboxyanhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine to generatea protected copolymer; treating the protected copolymer to partiallydepolymerize the protected copolymer, deprotect benzyl protected groupsand deprotect TFA-protected lysines to generate glatiramer acetate; andpurifying the glatiramer acetate, wherein the improvement comprises:measuring the amount of pyro-glutamate (pyro-Glu) in the purifiedglatiramer acetate. In other embodiments the improvement furthercomprises selecting the purified glatiramer acetate for use in thepreparation of a pharmaceutical composition if the amount of pyro-Glu inthe purified glatiramer acetate is within a predetermined range. In someembodiments the concentration of pyro-Glu in the selected purifiedglatiramer acetate 2000-7000 ppm or 2500-6500 ppm.

Also described is a method for preparing a pharmaceutical compositioncomprising glatiramer acetate, the method comprising: polymerizingN-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine to generatea protected copolymer; treating the protected copolymer to partiallydepolymerize the protected copolymer and deprotect benzyl protectedgroups and deprotecting TFA-protected lysines to generate glatirameracetate; and purifying the glatiramer acetate, wherein the improvementcomprises: measuring the amount of pyro-glutamate (pyro-Glu) during orafter the polymerizing step.

Described herein is a method for preparing a pharmaceutical compositioncomprising glatiramer acetate, the method comprising: polymerizingN-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine to generatea protected copolymer; treating the protected copolymer to partiallydepolymerize the protected copolymer and deprotect benzyl protectedgroups and deprotecting TFA-protected lysines to generate glatirameracetate; and purifying the glatiramer acetate, wherein the improvementcomprises: measuring the amount of pyro-glutamate (pyro-Glu) during orafter the partial depolymerization step.

In the aforementioned methods for preparing a pharmaceutical compositionthe improvement can further comprise: measuring the amount ofpyro-glutamate (pyro-Glu) in the purified glatiramer acetate; selectingthe purified glatiramer acetate for use in the preparation of apharmaceutical composition if the amount of pyro-Glu in the purifiedglatiramer acetate is within a predetermined range; and preparing apharmaceutical composition comprising at least a portion of the selectedpurified glatiramer acetate. In various embodiments, concentration ofpyro-Glu in the selected purified glatiramer acetate is, for example,2000-7000 ppm or 2500-6500 ppm.

Also described is a method for preparing a pharmaceutical compositioncomprising glatiramer acetate, comprising: polymerizing N-carboxyanhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine to generatea protected copolymer; treating the protected copolymer to partiallydepolymerize the protected copolymerand deprotect benzyl protectedgroups and deprotecting TFA-protected lysines to generate glatirameracetate; and purifying the glatiramer acetate, wherein the improvementcomprises: measuring the amount of benzyl alcohol during or after thepolymerizing step, wherein the amount of benzyl alcohol is correlated tothe amount of pyro-Glu.

Described herein is a method for preparing a pharmaceutical compositioncomprising glatiramer acetate, comprising: polymerizing N-carboxyanhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine to generatea protected copolymer; treating the protected copolymer to partiallydepolymerize the protected copolymer and deprotect benzyl protectedgroups and deprotecting TFA-protected lysines to generate glatirameracetate; and purifying the glatiramer acetate, wherein the improvementcomprises: measuring the amount of benzyl alcohol during or after thepartial depolymerization step, wherein the amount of benzyl alcohol iscorrelated to the amount of pyro-Glu.

In either of the methods entailing measuring the amount of benzylalcohol, the improvement can further comprise: measuring the amount ofpyro-glutamate (pyro-Glu) in the purified glatiramer acetate; selectingthe purified glatiramer acetate for use in the preparation of apharmaceutical composition if the amount of pyro-Glu in the purifiedglatiramer acetate is within a predetermined range; and preparing apharmaceutical composition comprising at least a portion of the selectedpurified glatiramer acetate. In various embodiments, the concentrationof pyro-Glu in the selected purified glatiramer acetate is, for example,2000-7000 ppm or 2500-6500 ppm.

The step of measuring the amount of pyro-Glu in a batch or sample caninclude any method for measuring (qualitatively or quantitatively) theamount of pyro-Glu and can include multiple steps and processes. Thus,the measuring step can include, for example: direct measurement of thecopolymer, size fractionating the copolymer, digesting the copolymer, orcleaving the copolymer. The measuring can be based on, for example, thetotal amount of pyro-Glu or on the concentration of pyro-Glu or on thepercentage of copolymer peptides that include a pyro-Glu. The measuredamount can be expressed in any convenient units, e.g., in weight, weightpercent or ppm (all measured in dry weight, i.e., total dry weightpyro-Glu in the sample/total dry weight of the sample), or mole percentof peptide chains. It should be noted that as the mole percent of chainsand weight percent of chains are related by the average molecular weightof the copolymer, it is possible to interconvert between these values ifthe average molecular weight is known, estimated or assumed. However,the precise value of the calculated mole percent of chains will dependon whether the average molecular weight value used is a number averagemolecular weight (Mn), weight average molecular weight (Mw) or peakaverage molecular weight (Mp). While Mw, Mp or Mn can be used in thecalculations, it is preferable to use Mn. Whatever method is used tomeasure pyro-Glu in the batch or sample, and whatever units are used toexpress the measured pyro-Glu in the batch or sample, the concentrationof pyro-Glu in the selected batch is between 2000 and 7000 ppm(mass_(pyro-Glu)mass_(total))×10⁶).

The methods can also include selecting the batch or pharmaceuticalpreparation as suitable for sale or administration to a human when theconcentration of pyro-Glu in the batch is within a predetermined range,e.g., 2000-7000 ppm.

The measuring step can comprise providing a value (e.g., in units suchas ppm, percent of peptide chains) for the amount of pyro-Glu in thebatch and optionally comparing the value to a reference value (e.g., aspecification for commercial release of a copolymer product).

Where the value for the amount of pyro-Glu in a batch of glatirameracetate has a preselected relationship with the reference value, themethod can include classifying, selecting, accepting, discarding,releasing, or withholding a batch of glatiramer acetate; reprocessing abatch through a previous manufacturing step; processing a batch ofglatiramer acetate into drug product, shipping the product from a batchof glatiramer acetate, moving the batch of glatiramer acetate to a newlocation; or formulating, labeling, packaging, selling, offering forsell, or releasing a batch of glatiramer acetate into commerce.

Also described herein is a method of analyzing a composition comprisingglatiramer acetate for the presence or amount of pyro-Glu, the methodcomprising: digesting a sample of the composition with a peptidase orprotease (e.g., pyroglutamate amino peptidase, an endopeptidase, andtrypsin), comparing the digestion products to a pyro-Glu referencestandard, and evaluating the amount of pyro-Glu in the sample relativeto the reference standard, thereby analyzing a composition comprisingglatiramer acetate. In some cases the digestion products are separatedby a chromatographic process prior to comparing the digestion to apyro-Glu reference standard. Thus, the comparison step can include achromatographic method (e.g., liquid chromatography, particularly HPLC)to separate components and mass spectrometry (MS) analysis or UVabsorbance analysis to detect the amount of various components.

In some cases the step of measuring pyro-Glu in the batch comprises:digesting a sample with a peptidase or a protease; isolating pyro-Glupresent in the digested sample; and measuring the amount of isolatedpyro-Glu. The isolating step can comprise a chromatographic method(e.g., liquid chromatography, particularly HPLC). The measuring step cancomprise mass spectrometry (MS) analysis or UV absorbance analysis

The measuring step can comprise measuring UV absorbance (e.g., at180-250 nm, 200 nm, or 210 nm). The isolating step can comprise achromatographic method (e.g., liquid chromatography, particularly HPLC).The determining step can comprise mass spectrometry (MS) analysis. Theisolating step can comprise HPLC and the measuring step can comprise UVabsorbance analysis. The isolating step can comprise liquidchromatography and the measuring step can comprise mass spectrometry(MS) analysis.

In some cases, the pyro-Glu content of the copolymer or glatirameracetate preparation is between 2000 to 7000 ppm, e.g., between 2500-6500ppm, e.g., between 3000-6000 ppm, e.g., between 3300-4400 ppm. In somecases, the pyro-Glu content of the copolymer or glatiramer acetatepreparation is less than 7000 ppm, e.g., less than 6000 ppm, less than5000 ppm, less than 4000 ppm, less than 3000 ppm, or less than 2000 ppm.

As used herein, a “copolymer”, “amino acid copolymer” or “amino acidcopolymer preparation” is a heterogeneous mixture of polypeptidescomprising a defined plurality of different amino acids (typicallybetween 2-10, e.g., between 3-6, different amino acids). A copolymer maybe prepared from the polymerization of individual amino acids. The term“amino acid” is not limited to naturally occurring amino acids, but caninclude amino acid derivatives and/or amino acid analogs. For example,in an amino acid copolymer comprising tyrosine amino acids, one or moreof the amino acids can be a homotyrosine. Further, an amino acidcopolymer having one or more non-peptide or peptidomimetic bonds betweentwo adjacent residues is included within this definition. A copolymer isnon-uniform with respect to the molecular weight of each species ofpolypeptide within the mixture.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows release of alanine from dipeptides upon HBr/acetic acidtreatment. A=Ala=Alanine; E=Glutamic Acid; K=Lysine; Y=Tyrosine. Alldipeptides were prepared at a concentration of 10 mM. Two dipeptides(A-A-NH2 and A-Y—NH2) were amidated at the C-terminus.

FIG. 2 is an LC-MS trace showing an unusual amino acid with residualmass of 111 Da (“X”) at the N-terminus of a peptide derived fromtrypsin-digested Copaxone®. Lys=Lysine; Ala=Alanine.

FIG. 3 shows the structure of L-pyro Glutamic Acid (pyro-Glu) GlatiramerAcetate (GA).

DETAILED DESCRIPTION OF THE INVENTION

Other than molecular weight and amino acid composition, which arespecified in the approved label for the product, the label and otheravailable literature for Copaxone® does not provide detailed informationabout the physiochemical characteristics of the product. Based ondetailed characterization of the product and process kinetics, theinventors have unexpectedly found a signature component of GA,L-pyro-Glutamic Acid (pyro-Glu) GA, that can be evaluated to assess theGA manufacturing process and product quality. In particular, evaluationof pyro-Glu content can identify differences in materials that are notobserved by looking at molar mass and amino acid composition alone. Byevaluating the pyro-Glu content of a sample of a copolymer, e.g., GA,one can identify non-conforming copolymer compositions. Accordingly,pyro-Glu content can be used to evaluate product and process quality forGA.

The production of GA entails both polymerization of amino acids andpartial depolymerization of the resulting peptides. It has now beenfound that depolymerization is highly specific and non-stochastic andoccurs to a disproportionately high extent to the N-terminal side ofglutamate residues. Indirectly, this results in pyro-Glu GA as asignature structural characteristic of GA, surprisingly occurringprimarily as a consequence of depolymerization. Pyro-Glu is present inGA in a range of 2000-7000 ppm and can be assessed to identify orevaluate GA and its method of manufacture, and/or to evaluate thequality or suitability of a GA product for pharmaceutical use.

Methods for Manufacture of Glatiramer Acetate

Generally, the process for the manufacture of glatiramer acetateincludes three steps:

Step (1): polymerization of N-carboxy anhydrides of L-alanine,benzyl-protected L-glutamic acid, trifluoroacetic acid (TFA) protectedL-lysine and L-tyrosine (collectively referred to as NCAs) to result ina protected copolymer,

Step (2): depolymerization and benzyl deprotection of the protectedcopolymer using hydrobromic acid in acetic acid, and

Step (3): deprotection of the TFA-protected lysines on the productcopolymers followed by purification and drying of the isolated drugsubstance.

In Step (1) of the manufacturing method, the NCAs are co-polymerized ina predetermined ratio using diethylamine as an initiator. Uponconsumption of the NCA components, the reaction mixture is quenched inwater. The resulting protected polymer (Intermediate-1) is isolated anddried. In Step (2), the protected polymer (Intermediate-1) is treatedwith anhydrous 33% HBr in acetic acid (HBr/AcOH). This results in thecleavage of the benzyl protecting group on the glutamic acid as well ascleavage of peptide bonds throughout the polymer, resulting in apartially depolymerized product (Intermediate-2) with a reducedmolecular weight relative to the parent Intermediate-1 polymer. Afterthe reaction is quenched with cold water, the product polymer isisolated by filtration and washed with water. The Intermediate-2material is dried before proceeding to Step (3). In Step (3),Intermediate-2 is treated with aqueous piperidine to remove thetrifluoroacetyl group on the lysine. The resulting copolymer(Intermediate-3) is subsequently purified usingdiafiltration/ultrafiltration and the resulting acetate salt dried toproduce Glatiramer Acetate drug substance.

Methods for the manufacture of glatiramer acetate have been described inthe following publications: U.S. Pat. No. 3,849,550; WO 95/031990 and US2007-0021324.

Process Chemistry of Synthetic Method and Structural Characterization ofGA

By studying the polymerization/depolymerization chemistry using modelpeptide compounds to model the synthetic process for producing GA, theinventors have found that there are certain rules associated with thechemistry. By developing an understanding of these rules, it is apparentthat GA is not a stochastic, or random, mixture of peptides. Rather,there are certain attributes that are conserved from batch-to-batch andcan be measured in order to monitor and evaluate process and batchquality.

Specifically, study of the kinetics of the depolymerization step of theGA manufacturing process using model peptide compounds revealed thatStep 2 depolymerization occurs to disproportionately high levels on theN-terminal side of glutamate residues. In model compounds, the onlyappreciable cleavage was on the N-terminal side of glutamate residues(FIG. 1). In the manufacturing process of Glatiramer Acetate, cleavageoccurs at all residues, but with a bias towards the N-terminal side ofglutamate residues. Further, a modified amino acid, identified aspyro-glutamic acid (pyro-Glu), was found in tryptic peptides ofCopaxone® samples. Analysis of aliquots removed from thedepolymerization step at various time points and then further processedto produce GA revealed that the amount of pyro-Glu at amino terminiincreases as the depolymerization time increases. Thus, the level ofpyro-Glu in the final GA product is surprisingly primarily a consequenceof the depolymerization kinetics and is not accounted for solely by thepolymerization chemistry. From this understanding of the chemistry of GAsynthesis, and from characterization of the resulting product, it hasthus been discovered that pyro-Glu is a signature structuralcharacteristic of glatiramer acetate. The formation of pyro-Glu resultsfrom: (1) parameters relating to the polymerization reaction, as wellas, surprisingly and unexpectedly, (2) parameters related to thede-polymerization reaction. Accordingly, pyro-Glu can be evaluated andmonitored in the manufacture of GA (including in the final drugsubstance or drug product) in order to, e.g., (i) identify GA, (ii)assess the quality of GA (e.g., of a GA batch), and/or (iii) assess orconfirm the quality of the GA manufacturing process.

Methods of Measuring Pyro-Glu

Because pyro-Glu is formed during the GA manufacturing process, itspresence and level provide useful information regarding GA chemistry andproduct quality.

Certain methods are described herein for measuring pyro-Glu content in acomposition that includes GA. However, it is understood that othermethods to measure pyro-Glu can also be used.

One analytical method developed and described herein for the measurementof pyro-Glu content is based on enzymatic cleavage of an N-terminalpyroglutamate residue using pyroglutamate aminopeptidase (e.g., fromthermophilic archaebacteria, Pyrococcus furiosus). The amount ofpyro-Glu in the resulting enzymatic hydrolysate can be analyzed by asuitable technique, such as reverse phase liquid chromatography, todetermine the ppm or w/w % of pyro-Glu in a GA sample. This method doesnot require knowing the mean chain length or average molecular weight ofthe GA in the composition. Accordingly, ppm or w/w % of pyro-Glu is apreferred expression of the amount of pyro-Glu in a batch or a sample ofcopolymer, e.g., GA.

Various methods can be used to determine the percentage of peptidechains bearing pyro-Glu in a GA sample. A determination of mole % orpercent of chains bearing pyro-Glu requires a determination of averagemolecular size or mean chain length. Molecular size can be evaluatede.g., by SEC MALLS (size exclusion chromatography with multiple anglelaser light scattering). Mean chain length can be computed e.g., bylabeling (e.g., with a radioactive or fluorescent label) the free aminotermini with a molecule which can be directly quantified. One analyticalmethod developed and described herein for measuring the percentage ofpeptide chains bearing pyro-Glu involves combining quantitative Edmandegradation with enzymatic removal of pyro-Glu. Such an analysis canentail: 1) quantifying the N-terminal amino acids in a sample of GAbefore treatment to remove pyro-Glu; and 2) quantifying the N-terminalamino acids in a sample of GA after treatment to remove pyro-Glu.

EXAMPLES Example 1 Depolymerization Kinetics of Glatiramer AcetateMethod of Manufacture

To investigate the depolymerization kinetics, the reaction of variousdipeptide model compounds with HBr/AcOH was investigated. FIG. 1 showsrelease of alanine from dipeptides upon HBr/acetic acid treatment asperformed in Step 2 of the manufacturing process. All dipeptides wereprepared at a concentration of 10 mM. Two dipeptides (A-A-NH2 andA-Y—NH2) were amidated at the C-terminus. As shown in FIG. 1, release ofalanine was only observed for A-E(OBn), indicating that dipeptides withGlu(OBn) in the C-terminal position demonstrate the most cleavage overthe course of 24-48 h reaction times as compared to dipeptides withoutGlu in the C-terminal position. Thus, depolymerization occurs to anappreciable extent only on the N-terminal side of glutamate residues inthese model systems. In the actual manufacturing process for GlatiramerAcetate, cleavage occurs at all residues, but still shows a strong biasfor the N-terminal side of glutamate residues.

Example 2 Presence of N-Terminal Pyro-Glu Structures

Trypsin digestion of Copaxone® followed by LC-MS analysis identifiedexpected peptides containing each of the amino acids A, E, K and Y. Inaddition, unexpected peptides were also found. An unusual amino acid(m/z 111) with residual mass of 111 Da was observed at the N-terminus ofseveral such unexpected peptides derived from trypsin-digested Copaxone®(labeled as “X”, FIG. 2). From LC-MS/MS analysis it was determined thatthe unusual amino acid is pyro-Glu, formed by cycling of N-terminalglutamic acid to form pyro-glutamic acid losing a water molecule [111Da=129 Da (Glutamic acid residue)−18 Da (H2O)]. FIG. 3 shows thestructure of L-pyro Glutamic Acid (pyro-Glu) GA.

Example 3 Evaluation of Pyro-Glu Content on a Weight Basis

This example describes a method for evaluating pyro-Glu content in acopolymer composition.

An analytical method developed for the pyro-glutamate content assay isbased on enzymatic cleavage of a N-terminal pyro-glutamate residue usingpyro-glutamate aminopeptidase (from thermophilic archaebacteria,Pyrococcus furiosus). Pyro-glutamate in the resulting enzymatichydrolysate is isolated by reverse phase liquid chromatography followedby detection at 200 nm using a reference standard curve prepared withknown concentrations of L-Pyro-glutamate. Neurotensin (a commerciallyavailable polypeptide having 100% pyro-glutamate at the N-terminus) isassayed as a control to ensure the acceptability of the digestion andadequacy of the HPLC separation. The chromatographic analysis isperformed using a Waters Atlantis C18 HPLC column and an isocraticmobile phase consisting of 100% Water, adjusted to pH 2.1 withphosphoric acid. Samples and Standards are held at 2-8° C. The peakcorresponding to the pyro-glutamate moiety elutes at a retention time ofapproximately 12 minutes. The direct measure of pyro-glutamate contentis on a w/w basis and the results are expressed as ppm (microgram/gram).

Example 4 Evaluation of Pyro-Glu Content on a Percentage of PeptideChains Basis

The percentage of peptide chains in a sample of GA bearing pyro-Glu canbe measured as an alternative to measuring the amount of pyro-Glu in asample of GA. The percentage of peptide chains bearing pyro-Glu can bedetermined by combining quantitative Edman degradation with enzymaticremoval of pyro-Glu. Thus, the analysis entails: 1) quantifying theN-terminal amino acids in a sample of GA before treatment to removepyro-Glu; and 2) quantifying the N-terminal amino acids in a sample ofGA after treatment to remove pyro-Glu.

An Edman degradation reaction was used to quantify the N-terminal aminoacids in a sample of GA before and after treatment with pyroglutamateaminopeptidase (PA) to remove pyro-Glu. This reaction was performedmanually to avoid quantitative limitations of automatic N-terminalpeptide sequencers. The results of this analysis are presented in thetable below.

TABLE 1 N-terminal Amino Acid nmol N-terminal amino acid Before PATreatment After PA Treatment Amino Acid (st. dev) (st. dev) Ala 25.1(0.6)  51.7 (0.5) Glu 7.8 (0.3) 15.7 (0.1) Lys 9.0 (0.2) 20.2 (0.8) Tyr6.5 (0.1) 10.5 (0.2) Total 48.4 98.1

As can be seen in Table 1, above, the N-terminal amino acidconcentration increased from 48.4 to 98.1 nmol after PA treatment. Thisis because removal of pyro-Glu permits detection of peptides that couldnot previously have been detected by Edman degradation. The percentageof chains bearing pyro-Glu can be calculated as follows: % chains cappedby pyroglutamate=(Pafter−Pbefore)/Pafter×100%. In this calculation,Pbefore and Pafter are the concentrations of N-terminal amino acids withand without PA treatment, respectively. In this example, 51% of thepolymer chains were capped by pyroglutamate.

Example 5 Pyro-Glu Content can Distinguish Glatiramer Acetate

Using the method described in Example 3, the pyro-Glu content ofcommercial Copaxone® was compared to several other copolymer samples. Asample of glatiramer acetate (M-GA) prepared according to the methoddescribed in U.S. Pat. No. 3,849,550 was evaluated for pyro-Glu content.Table 2, below, provides the results of the analysis of a number ofcompositions, this sample conforms to the range found for pyro-Glucontent from a sampling of Copaxone® lots, or between 2500-6500 ppm.

TABLE 2 Analysis of Samples Analysis of Samples Molecular weight Aminoacid composition P-Glu content Sample (Mp) (Da) (avg. molar fraction)²(ppm) Copaxone ® 5,000-9,000¹ 0.141 L-Glutamic acid 2500-6500 ppm⁴ 0.427L-alanine 0.095 L-tyrosine 0.338 L-lysine Glatiramer 8407 (conforms)³4900 ppm acetate (conforms) (conforms) sample (M-GA) Deviating 6579(conforms)³ 8200 ppm sample A (conforms) (fails) Deviating 4808(conforms)³ 7500 ppm sample B (fails) (fails) ¹Molecular weight rangespecified in Copaxone ® product label and prescription information²Average molar fraction target specified in Copaxone ® product label andprescription information ³Conforms relative to specification range basedon label target plus allowance for manufacturing and measurementvariability ⁴Range is 75%/125% of Copaxone min/max for 30 commercialsamples

To test the ability of pyro-Glu content to distinguish glatirameracetate from non-conforming copolymers, two control copolymers weretested. The control copolymers were made with deliberate and specificdeviations in the timing of NCA addition or in the duration of step 2.As shown in Table 1, both deviating samples A and B were outside of therange for pyro-Glu content determined for Copaxone®. Sample A was withinthe range for Copaxone® molar mass and amino acid composition whileSample B failed molar mass but conformed in amino acid composition. Thisdata shows that evaluation of pyro-Glu content can identify differencesin materials and process not observed by looking at molar mass and aminoacid composition alone and illustrates the ability of pyro-Glumeasurement to identify non-conforming copolymer. Accordingly, pyro-Glucontent can be used to evaluate product and process quality forglatiramer acetate.

1. (canceled)
 2. A method for manufacturing glatiramer acetate, themethod comprising: preparing an amino acid copolymer of L-glutamic acid,L-alanine, L-lysine, and L-tyrosine; measuring pyro-glutamate content ofthe copolymer in a sample of the copolymer; processing the copolymer toproduce glatiramer acetate if the measured pyro-glutamate content of thecopolymer in the sample is within 2000-7000 parts per million (ppm) on adry weight/dry weight basis.
 3. The method of claim 2 wherein: the stepof preparing comprises co-polymerizing N-carboxy anhydrides ofL-alanine, benzyl-protected L-glutamic acid, trifluoroacetic acid(TFA)-protected L-lysine, and L-tyrosine to generate a first material;treating the first material to deprotect the benzyl-protected L-glutamicacid therein and to partially depolymerize the first material, therebygenerating a second material; and purifying and optionally drying thesecond material; or purifying and optionally drying the second materialto produce a third material and treating the third material to deprotectthe TFA-protected L-lysine, to thereby produce the copolymer ofL-glutamic acid, L-alanine, L-lysine, and L-tyrosine.
 4. The method ofclaim 3, wherein the processing step comprises processing the copolymerto produce glatiramer acetate drug substance if the measuredpyro-glutamate content of the copolymer in the sample is within2000-7000 ppm on a dry weight/dry weight basis.
 5. The method of claim4, further comprising processing the glatiramer acetate drug substanceto produce glatiramer acetate drug product.
 6. The method of claim 2,wherein: the step of preparing comprises co-polymerizing N-carboxyanhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA)-protected L-lysine, and L-tyrosine togenerate a first material; treating the first material to deprotect thebenzyl-protected L-glutamic acid therein and to partially depolymerizethe first material, thereby generating a second material; purifying anddrying the second material to produce a purified and dried secondmaterial; treating the purified and dried second material to deprotectthe TFA-protected L-lysine to produce a third material; and purifyingand optionally drying the third material, to thereby produce thecopolymer of L-glutamic acid, L-alanine, L-lysine, and L-tyrosine. 7.The method of claim 6, comprising purifying and drying the thirdmaterial, to thereby produce the copolymer of L-glutamic acid,L-alanine, L-lysine, and L-tyrosine.
 8. The method of claim 6, whereinthe processing step comprises processing the copolymer to produceglatiramer acetate drug product if the measured pyro-glutamate contentof the copolymer in the sample is within 2000-7000 ppm on a dryweight/dry weight basis.
 9. The method of claim 7, wherein theprocessing step comprises processing the copolymer to produce glatirameracetate drug product if the measured pyro-glutamate content of thecopolymer in the sample is within 2000-7000 ppm on a dry weight/dryweight basis.
 10. The method of claim 6, wherein purifying and dryingthe third material comprises diafiltration/ultrafiltration.
 11. Themethod of claim 7, wherein purifying and drying the third materialcomprises diafiltration/ultrafiltration.
 12. The method of claim 2,wherein measuring pyro-glutamate content of the copolymer in a sample ofthe copolymer comprises obtaining a sample of a manufacturing batch ofthe copolymer.
 13. The method of any claim 2, wherein measuringpyro-glutamate content of the copolymer in a sample of the copolymercomprises digesting the copolymer in the sample with pyroglutamateaminopeptidase.
 14. The method of any one of claim 2, wherein measuringpyro-glutamate content comprises determining the ppm on a dry weight/dryweight basis of gyro-Glu in the sample of the copolymer.
 15. The methodof claim 2, further comprising packaging, labeling, or releasing intocommerce the glatiramer acetate.
 16. The method of claim 2, furthercomprising offering for sale or selling the glatiramer acetate.
 17. Themethod of one of claim 2, further comprising shipping or moving to a newlocation the glatiramer acetate.
 18. The method of claim 3, whereinco-polymerizing N-carboxy anhydrides of L-alanine, benzyl-protectedL-glutamic acid, trifluoroacetic acid (TFA)-protected L-lysine, andL-tyrosine to generate a first material comprises contacting theN-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA)-protected L-lysine, and L-tyrosine withdiethylamine; and wherein treating the third material to deprotect theTFA-protected L-lysine comprises treating with aqueous piperidine. 19.The method of claim 6, wherein co-polymerizing N-carboxy anhydrides ofL-alanine, benzyl-protected L-glutamic acid, trifluoroacetic acid(TFA)-protected L-lysine, and L-tyrosine to generate a first materialcomprises contacting the N-carboxy anhydrides of L-alanine,benzyl-protected L-glutamic acid, trifluoroacetic acid (TFA)-protectedL-lysine, and L-tyrosine with diethylamine; and wherein treating thethird material to deprotect the TFA-protected L-lysine comprisestreating with aqueous piperidine.
 20. The method of claim 3, whereintreating the first material to deprotect the benzyl-protected L-glutamicacid therein and to partially depolymerize the first material comprisesthe first material with phenol treated HBr/acetic acid.
 21. The methodof claim 6, wherein treating the first material to deprotect thebenzyl-protected L-glutamic acid therein and to partially depolymerizethe first material comprises the first material with phenol treatedHBr/acetic acid.
 22. The method of claim 2, wherein the glatirameracetate has an Mp of 5000-9000 Da.
 23. A method for manufacturingglatiramer acetate, the method comprising: preparing glatiramer acetatedrug product; measuring pyro-glutamate content of the drug product in asample of the drug product; and shipping or moving to a new location thedrug product if the measured pyro-glutamate content of the drug productin the sample is within 2000-7000 parts per million (ppm) on a dryweight/dry weight basis.
 24. The method of claim 23, further comprisingoffering for sale or selling the drug product.
 25. The method of claim23, wherein the step of preparing comprises co-polymerizing N-carboxyanhydrides of L-alanine, benzyl-protected L-glutamic acid,trifluoroacetic acid (TFA)-protected L-lysine, and L-tyrosine togenerate a first material; treating the first material to deprotect thebenzyl-protected L-glutamic acid therein and to partially depolymerizethe first material, thereby generating a second material; purifying anddrying the second material to produce a purified and dried secondmaterial; treating the purified and dried second material with aqueouspiperidine to deprotect the TFA-protected L-lysine to produce a thirdmaterial; purifying and drying the third material, to produce glatirameracetate drug substance; and processing the glatiramer acetate drugsubstance to prepare glatiramer acetate drug product.
 26. The method ofclaim 25, wherein purifying and drying the third material comprisesdiafiltration/ultrafiltration.
 27. The method of claim 23, whereinmeasuring pyro-glutamate content comprises digesting the drug product inthe sample with pyroglutamate aminopeptidase.
 28. The method of claim25, wherein measuring pyro-glutamate content comprises digesting thedrug product in the sample with pyroglutamate aminopeptidase.
 29. Themethod of claim 23, wherein measuring pyro-glutamate content comprisesdetermining the ppm on a dry weight/dry weight basis of pyro-Glu in thesample of the copolymer.
 30. The method of claim 25, wherein measuringpyro-glutamate content comprises determining the ppm on a dry weight/dryweight basis of pyro-Glu in the sample of the copolymer.
 31. The methodof claim 23, wherein the glatiramer acetate drug product has an Mp of5000-9000 Da.